The role of cytosolic DNA sensors in colitis and intestinal cancer

Abstract

Background and Aim: The innate immune response contributes to the development or attenuation of cancer. In the gastrointestinal tract, DNA is often liberated from microbes or damaged host cells and can activate protective responses against the development of cancer. Patients with colorectal cancer whose tissues express a higher level of the DNA sensor Ku70 survive longer than those with a lower expression. In addition, the gene encoding Ku70 is frequently mutated in tumour samples from patients with colorectal cancer, suggesting a putative role of Ku70 in the development and/or progression of colorectal cancer. Ku70 has an established role in DNA repair and has been implicated in controlling intestinal homeostasis in mice. This PhD thesis investigates the role of Ku70 in colitis-associated colorectal cancer and genetically-predisposed intestinal cancer in mice and characterises the mechanisms of action of the Ku70 signalling pathway in tumorigenesis. Methods: Two mouse models of intestinal cancer are used. In the colitis and colitis-associated colorectal cancer model, littermate wild-type (WT) mice and mice lacking one allele of Ku70 (Ku70-Het mice) or both alleles of Ku70 (Ku70-KO mice) were treated with the procarcinogen azoxymethane and the inflammatory agent dextran sodium sulfate. In the genetically-predisposed intestinal cancer model, WT and Ku70-Het mice were crossed with Apc-Het mice carrying a mutation in the gene encoding the tumour suppressor protein APC, which predisposes mice to the development of spontaneous intestinal cancer. In both models, tumour numbers were enumerated in the intestinal tissue of mice. To identify the signalling pathways controlled by Ku70, I performed a phosphoproteomic screen using mass spectrometry. Further, I used histology, immunohistochemistry, immunoblotting, ELISA, qRT-PCR, overexpression system and immunoprecipitation techniques to gain insights into the molecular changes during the development of colitis and intestinal cancer. Results: I found that Ku70-Het mice lost more body weight during the development of colitis and colitis-associated colorectal cancer and had a greater tumour burden compared with littermate WT mice. Ku70-KO mice were highly susceptible to colitis and did not survive the duration of the colitis-associated colorectal cancer experiment. Furthermore, Apc-Het-Ku70-Het mice had enhanced tumour burden in the small and large intestine compared to littermate Apc-Het mice. The effects of Ku70 on attenuating colitis and intestinal cancer were independent of the role of Ku70 in DNA repair, and the production of inflammatory markers and interferons. Instead, Ku70 interacted with the GTPase Ras and the kinase Raf, forming a tripartite protein complex that activated the kinases MEK and ERK. I show that the alpha/beta-domain and the C-terminal domain of Ku70 interacted with the GTPase domain of Ras, whereas the C-terminal domain of Ku70 interacted with the conserved region 2 of Raf. The Ku70-Ras-Raf complex induced MEK-ERK activation, leading to reduced activation of the phosphatase Cdc25A and cell-cycle kinase CDK1, and reduced tumour burden. The Ku70-Ras-Raf complex interacted with endosomal markers Rab5 and Rab7, suggesting possible docking at the endosomal membrane. Moreover, my preliminary data indicate that Ku70 may migrate from the nucleus to the cytoplasm during the development of colitis, presumably for the sensing of cytoplasmic DNA. Conclusions: In this thesis, I uncovered that Ku70 induces the formation of a novel signalosome complex, composed of Ku70, Ras and Raf. This signalosome docks at the endosomal membrane and induces activation of the MEK-ERK signalling pathway during tumorigenesis, potentially attenuating the development of intestinal cancer. These findings may facilitate the design of therapeutics targeting components of the Ku70 signallin